Effect of peppermint (Mentha piperita) oil on in vitro methanogenesis and fermentation of feed with buffalo rumen liquor

The effect of inclusion of peppermint (Mentha piperita) oil (at 0, 0.33, 1.0 and 2.0 μl/ml of incubation medium) on gas and methane production, fermentation of feed and microbial profile was studied in in vitro gas production test, using 200 mg of wheat straw and concentrate mixture in equal proportion as substrate in a 100 ml graduated syringe. The buffalo rumen liquor was used as inoculum and the observations were recorded at 24 h of incubation. Methane emission was reduced (P<0.001) by 19.9%, 46.0% and 75.6% at 0.33, 1.0 and 2.0 μl levels, respectively. The concentration (mM/100 ml) of total volatile fatty acids was reduced (P<0.01) by inclusion of peppermint oil at higher levels (1.0 and 2.0 μl) whereas at 0.33 μl level there was no effect. The proportion of acetate increased (P<0.05) and that of propionate decreased (P<0.001) at 1.0 and 2.0 μl levels of peppermint oil. There was a fall (P<0.001) in carboxymethylcellulase and xylanase activities and the inhibition increased with the increasing level of peppermint oil which resulted in a dose dependent decrease (P<0.05) in in vitro true digestibility of feed. At 0.33 μl level of peppermint oil, the population density of total bacteria was similar to that of control but fungi, Ruminococcus flavefaciens and methanogens increased by 4-, 6- and 2-folds, respectively, as determined with real-time PCR. At 1.0 and 2.0 μl levels the population density of total bacteria, fungi, Fibrobacter succinogens and methanogens decreased drastically and fell below the control values. The numbers of holotrichs and spirotrichs were reduced (P<0.001) by increasing dose of peppermint oil. The higher doses of peppermint oil were toxic for the rumen microbes but the lower levels could be further explored in in vivo experiments as rumen modifier to reduce methanogenesis.     

On-line detection of low naphthalene concentrations with a bioluminescent sensor

On-line tests with Pseudomonas fluorescens HK44 were carried out using a biosensor system with a flow-through cell and sample injections to detect low concentrations of naphthalene. Kinetic experiments permitted a 19 min response time and definition of a 50 min induction period for bioluminescence assays. A mathematical model showed that 0.2 g/l is the best cell concentration to be used in the detection of low naphthalene concentrations, with the highest acceleration rate of bioluminescence sensitivity increase (0.46 nA l/(g min²)). Calibration curves with different concentrations of naphthalene showed a linear correlation up to 0.4 mg/l (3 μM) corresponding to 211 nA output signal. The lower limit of naphthalene detection by strain HK44 was in the region of 0.02 mg/l (0.16 μM), below the naphthalene health advisory limit for drinking water consumed over a lifetime suggested by EPA.     

Influences of flavomycin,ropadiar, and saponin on nutrient digestibility,rumen fermentation,and methane emission from sheep

This study focused on the effects of three additives given together with a hay/concentrate-based diet on nutrient digestibility, rumen fermentation, and methane emission from sheep. The basal diet consisted of 1.29 kg mixed hay and 0.43 kg concentrate mixture based on dry matter (DM). Treatments consisted of control (no additive), flavomycin⁴⁰ (250 mg/d), ropadiar from an oregano extract (250 mg/d), and saponin in the form of a yucca schidigera extract (170 mg/d). Results indicated that intake and digestibility were unaffected by treatments (P>0.05). The NH₃-N concentration of rumen liquor was lower (P<0.05) for additive treatments versus the control treatment. Higher concentrations of volatile fatty acid (VFA) were observed in the saponin (75.8 mmol/L) and ropadiar (73.1 mmol/L) treatments. The proportion of individual fatty acid of rumen liquor was unchanged, whereas lower ratio of acetate to propionate in the saponin treatment was observed (P<0.05). The average methane production expressed on digested organic matter (OM) and neutral detergent fiber (aNDFom) basis were decreased by approximately 3.3 and 12.0 g/kg, respectively in saponin, and 4.2 and 11.9 g/kg in ropadiar treatment compared to the control. Methane production was positively correlated with the concentrations of NH₃-N, and negatively correlated with total VFA and the proportion of propionate of rumen liquor (P<0.05). The study found that saponin and ropadiar could have the potential to reduce rumen methanogenesis in sheep.     

The relationship between the volatile fatty acids supply and the nitrogen retention in growing sheep nourished by total intragastric infusions

Twelve four-month old Suffolk × Small-tail-Han male sheep (live weight 21–26 kg), fitted with rumen and abomasum fistulas and nourished by total intragastric infusions, were used to study the relationship between the volatile fatty acids (VFA) supply and the nitrogen (N) retention in sheep. The animals were randomly divided into four groups and four levels of mixed VFA energy (the molar proportion of acetic acid, propionic acid and butyric acid was 65:25:10), i.e. 333, 378, 423 and 468 kJ kg^?1 W^0.75 d^?1, were infused into the rumen, as treatments I, II, III and IV, respectively. The results showed that the N retention was significantly increased (P < 0.05) with the VFA infusion level. Significant regression relationship was found between the VFA supply (x, g d^?1) and the N retention (y, mg d^?1): y = 2.75x ? 403, r² = 0.86, n = 12, P < 0.01. It was concluded that to get efficient utilization of dietary N and high N retention in sheep, it is necessary to supply enough dietary energy.     

Evaluation of insecticidal activity of the essential oil of Allium chinense G. Don and its major constituents against Liposcelis bostrychophila Badonnel

Water-distilled essential oil from the dried bulbs of Allium chinense (Liliaceae) was analyzed by gas chromatography–mass spectrometry (GC–MS). Eighteen compounds, accounting for 98.4% of the total oil, were identified and the main components of the essential oil of A. chinense were methyl allyl trisulfide (30.7%), dimethyl trisulfide (24.1%), methyl propyl disulfide (12.8%) and dimethyl disulfide (9.6%) followed by methyl allyl disulfide (3.4%) and methyl propyl trisulfide (3.6%). The essential oil exhibited contact toxicity against the booklice (Liposcelis bostrychophila) with an LC₅₀ value of 441.8 μg/cm² while the two major constituents, dimethyl trisulfide and methyl propyl disulfide had LC₅₀ values of 153.0 μg/cm² and 738.0 μg/cm² against the booklice, respectively. The essential oil of A. chinense possessed strong fumigant toxicity against the booklice with an LC₅₀ value of 186.5 μg/l while methyl allyl trisulfide (LC₅₀ = 90.4 μg/l) and dimethyl trisulfide (LC₅₀ = 114.2 μg/l) exhibited stronger fumigant toxicity than methyl propyl disulfide (LC₅₀ = 243.4 μg/l) and dimethyl disulfide (LC₅₀ = 340.8 μg/l) against the booklice. The results indicated that the essential oil and its major constituents have potential for development into natural insecticides or fumigants for control of insects in stored grains.     

Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C₁₈ 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.     

Effect of serum albumin supplementation on in vitro capacitation and fertilization of caprine oocytes

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO₂ in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes (n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes (n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes (n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes (n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher (P < 0.05) cleavage rate, compared to unmodified albumin, and the supplementation of 20% estrous goat serum in the fertilization medium, significantly (P < 0.05) increased the cleavage rate of in vitro matured goat oocytes, compared to defatted albumin.     

Continuous hydrolysis of carboxymethyl cellulose with cellulase aggregates trapped inside membranes

Enzymatic hydrolysis of cellulose is often conducted in batch processes in which hydrolytic products tend to inhibit enzyme activity. In this study, we report a method for continuous hydrolysis of carboxymethyl cellulose (CMC) by using cross-linked cellulase aggregate (XCA) trapped inside a membrane. XCA particles prepared by using a millifluidic reactor have a uniform size distribution around 350 nm. Because of their large size, XCA particles in solutions can be filtered through a polyethersulfone membrane to collect 87.1 ± 0.9% of XCA particles. The membrane with impregnated XCA can be used as a catalyst for hydrolysis of CMC in a continuous mode. When the CMC concentration is 1.0 g/l and the flow rate is 2 μl/min, 53.9% of CMC is hydrolyzed to reducing sugars. The membrane with XCA is very stable under continuously flowing solutions. After 72 h of reaction, 97.5% of XCA remains inside the membrane.     

Effects of dose and adaptation time of a specific blend of essential oil compounds on rumen fermentation

Essential oils can be used as natural additives in animal feeds. The present study evaluated the effects of three different doses and different adaptation times of a specific blend of essential oils (BEO) on rumen microbial fermentation. Eight dual flow continuous culture fermenters (1320 ml) were used in two periods of 9 days each to study the effects of increasing doses of BEO. Treatments were: CTR (no BEO), D5 (5 mg/l of BEO), D50 (50 mg/l of BEO) and D500 (500 mg/l of BEO). During the last 3 days, samples were taken at 0, 2, 4 and 6 h after the morning feeding and analyzed for large peptide (LPep), small peptides plus amino acid (SPep + AA) and ammonia N concentrations, and at 2 h after feeding for volatile fatty acids (VFA) concentration and profile. The D5 increased total VFA concentration, acetate proportion and acetate to propionate ratio, and decreased propionate and valerate proportion, compared with CTR. The concentration of LPep N tended (P=0.08) to be lower for D5 compared with CTR. In the second experiment, eight sheep were used to study the effects of long-term adaptation of rumen fluid to BEO on ruminal fermentation. Four sheep were assigned at random to the CTR treatment (no BEO) and four sheep were adapted to BEO (110 mg/day of BEO) for 4 weeks (ADBEO). After 4 weeks samples of ruminal fluid were obtained at 0 and 3 h after the morning feeding and in 2 consecutive days using an oro-ruminal probe. Samples were analyzed for LPep, SPep + AA and ammonia N concentrations, total and individual VFA, and pH. Treatment ADBEO tended (P<0.10) to increase acetate proportion and decrease valerate proportion, compared with CTR. Ruminal fluid collected from each of CTR and ADBEO sheep was used to study in vitro fermentation profile of soybean meal, corn meal, alfalfa hay and ryegrass hay. Treatments were: Control fluid (CTR without BEO), CTR fluid plus a single dose of BEO (11 mg/l; CTR + BEO) and ADBEO fluid plus a single dose of BEO (11 mg/l; ADBEO + BEO). Acetate proportion and acetate to propionate ratio was higher, and propionate and isovalerate proportion, and BCVFA and ammonia N concentration were lower in ADBEO + BEO fluid compared with CTR fluid. The addition of essential oils can shift the microbial fermentation in the rumen by increasing the acetate to propionate ratio and inhibiting deamination.     

Models of hemorrhagic shock: Differences in the physiological and inflammatory response

IntroductionThe hemorrhagic shock (HS) model is commonly used to initiate a systemic post-traumatic inflammatory response. Numerous experimental protocols exist and it is unclear how differences in these models affect the immune response making it difficult to compare results between studies. The aim of this study was to compare the inflammatory response of different established protocols for volume-controlled shock in a murine model.MethodsMale C57/BL6 mice 6–10 weeks and weighing 20–25 g were subjected to volume-controlled or pressure-controlled hemorrhagic shock. In the volume-controlled group 300 μl, 500 μl, or 700 μl blood was collected over 15 min and mean arterial pressure was continuously monitored during the period of shock. In the pressure-controlled hemorrhagic shock group, blood volume was depleted with a goal mean arterial pressure of 35 mmHg for 90 min. Following hemorrhage, mice from all groups were resuscitated with the extracted blood and an equal volume of lactated ringer solution. Six hours from the initiation of hemorrhagic shock, serum IL-6, KC, MCP-1 and MPO activity within the lung and liver tissue were assessed.ResultsIn the volume-controlled group, the mice were able to compensate the initial blood loss within 30 min. Approximately 800 μl of blood volume was removed to achieve a MAP of 35 mmHg (p < 0.001). No difference in the pro-inflammatory cytokine (IL-6 and KC) profile was measured between the volume-controlled groups (300 μl, 500 μl, or 700 μl). The pressure-controlled group demonstrated significantly higher cytokine levels (IL-6 and KC) than all volume-controlled groups. Pulmonary MPO activity increased with the severity of the HS (p < 0.05). This relationship could not be observed in the liver.ConclusionVolume-controlled hemorrhagic shock performed following current literature recommendations may be insufficient to produce a profound post-traumatic inflammatory response. A decrease in the MAP following blood withdrawal (300 μl, 500 μl or 700 μl) was usually compensated within 30 min. Pressure-controlled hemorrhagic shock is a more reliable for induction of a systemic inflammatory response.     

Environmental stresses induce health-promoting phytochemicals in lettuce

Plants typically respond to environmental stresses by inducing antioxidants as a defense mechanism. As a number of these are also phytochemicals with health-promoting qualities in the human diet, we have used mild environmental stresses to enhance the phytochemical content of lettuce, a common leafy vegetable. Five-week-old lettuce (Lactuca sativa L.) plants grown in growth chambers were exposed to mild stresses such as heat shock (40 °C for 10 min), chilling (4 °C for 1 d) or high light intensity (800 μmol m^?2 s^?1 for 1 d). In response to these stresses, there was a two to threefold increase in the total phenolic content and a significant increase in the antioxidant capacity. The concentrations of two major phenolic compounds in lettuce, chicoric acid and chlorogenic acid, increased significantly in response to all the stresses. Quercetin-3-O-glucoside and luteolin-7-O-glucoside were not detected in the control plants, but showed marked accumulations following the stress treatments. The results suggest that certain phenolic compounds can be induced in lettuce by environmental stresses. Of all the stress treatments, high light produced the greatest accumulation of phenolic compounds, especially following the stress treatments during the recovery. In addition, key genes such as phenylalanine ammonia-lyase (PAL), l-galactose dehydrogenase (l-GalDH), and γ-tocopherol methyltransferase (γ-TMT) involved in the biosynthesis of phenolic compounds, ascorbic acid, and α-tocopherol, respectively, were rapidly activated by chilling stress while heat shock and high light did not appear to have an effect on the expression of PAL and γ-TMT. However, l-GalDH was consistently activated in response to all the stresses. The results also show that these mild environmental stresses had no adverse effects on the overall growth of lettuce, suggesting that it is possible to use mild environmental stresses to successfully improve the phytochemical content and hence the health-promoting quality of lettuce with little or no adverse effect on its growth or yield.     

Effects of phosphorus and nitrogen amendments on the growth of Egeria najas

The effect of nutrient addition on the growth of E. najas was evaluated in a dose response experiment using sand amended with phosphorus (P) and nitrogen (N), and in enrichment trials with N and P amendments to natural sediments. Plants, water and sediment came from lagoons of the Upper Paraná River Floodplain and from Itaipu Reservoir (Brazil). Relative growth rates (RGRs) of E. najas shoots, based on dry mass (DM), varied from 0.03 to 0.060 d⁻¹ for both nutrients. Root:shoot biomass ratios were related to sediment exchangeable P (r = −0.419; P = 0.03) and N (r = −0.54; P = 0.006), however root RGR was not related to sediment nutrient concentrations. When natural sediments were amended with N and P, neither shoot nor root RGRs differed among treatments for substrata from either the reservoir or the floodplain lagoons (P > 0.05). Comparison of nutrient concentrations measured in natural sediments collected from several sites in both the Upper Paraná River Floodplain (range 49–213 μg P g⁻¹ DM; 36–373 μg N g⁻¹ DM) and Itaipu Reservoir (range 43–402 μg P g⁻¹ DM; 7.9–238 μg N g⁻¹ DM) showed that sediment N and P from these systems usually exceeded minimum requirements necessary for E. najas growth, as measured in the dose response experiment. Together, these results indicate that E. najas, at least in early stages of development, responds to sediment nutrient amendments and relies upon bottom sediments to meet its N and P requirements and that for at least two Brazilian ecosystems, growth of this species is not limited by insufficient sediment N or P. Thus, reducing N and P in water is not enough to control E. najas growth in short time periods in these ecosystems.     

Immune responses,ROS generation and the haemocyte damage of scallop Chlamys farreri exposed to Aroclor 1254

The toxic effects of Aroclor 1254 (0.05, 0.5, 5 and 50 μg l^?1) on scallop (Chlamys farreri) immune system in vivo were studied. The results showed that Aroclor 1254 had significant toxic effect on the parameters tested in this paper (P < 0.05). The total number of haemocytes, the proportion of granulocytes, phagocytosis in all groups as well as the lysosomal membrane stability (LMS) in 5, 50 μg l^?1 and bacteriolytic activity 0.5, 5, 50 μg l^?1 treatments decreased significantly, while the proportion of hyalinocytes and the production of O₂^- in all treatments remarkably increased during the sampling time and tended to be stable gradually after 6–15 d. The bacteriolytic activity in 0.05 μg l^?1 treatments, LMS in 0.05, 0.5 μg l^?1 groups and the DNA damage (comet ratios and arbitrary values) in all treatments increased at the beginning of exposure and reached their peaks on day 1, day 1, day 6 and day 3, following that they all decreased gradually and became stable after 9–15 d. When the indices reached stability, except for DNA damage was higher than controls, the others were all significantly lower than those of controls (P < 0.05). Thus, Aroclor 1254 has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms. Also it supports the speculation that the PCBs pollution is one of the important reasons of the mass mortality of the C. farreri.     

The effects of clove oil on the enzyme activity of Varroa destructor Anderson and Trueman (Arachnida: Acari: Varroidae)

Varroa destructor, a key biotic threat to the Western honey bee, has played a major role in colony losses over the past few years worldwide. Overuse of traditional acaricides, such as tau-fluvalinate and flumethrin, on V. destructor has only increased its tolerance to them. Therefore, the application of essential oils in place of traditional pesticides is an attractive alternative, as demonstrated by its high efficiency, lack of residue and tolerance resistance. To study the acaricidal activity of essential oils, we used clove oil (Syzygium aromaticum L.), a typical essential oil with a wide range of field applications, and examined its effects on the enzyme activities of Ca²⁺-Mg²⁺-ATPase, glutathione-S-transferase (GST) and superoxide dismutase (SOD) and its effects on the water-soluble protein content of V. destructor body extracts after exposure to 0.1 μl and 1.0 μl of clove oil for 30 min. Our results showed that the water-soluble protein content significantly decreased after the treatments, indicating that the metabolism of the mites was adversely affected. The bioactivity of GSTs increased significantly after a low dosage (0.1 μl) exposure but decreased at a higher dosage (1.0 μl), while the activities of SOD and Ca²⁺-Mg²⁺-ATPase were significantly elevated after treatments. These results suggest that the protective enzyme SOD and detoxifying enzymes Ca²⁺-Mg²⁺-ATPase and GST contributed to the stress reaction of V. destructor to the essential oils and that the detoxification ability of V. destructor via GST was inhibited at higher dosages. Our findings are conducive to understanding the physiological reactions of V. destructor to treatment with essential oils and the underlying mechanisms behind the acaricidal activities of these natural products.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 °C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 μM BCB. There were fewer BCB+ oocytes after exposure to 13 μM BCB (10%) than after exposure to 26 or 39 μM BCB (57.2 and 61.8%; P < 0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4 ± 4.2 μm; mean ± S.E.M.) was higher (P < 0.05) than that of BCB− oocytes (136.8 ± 4.6 μm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 μM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P < 0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB− (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P < 0.05); blastocyst development was lowest in BCB− oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

Cultivation impacts soil microbial dynamics in dry tropical forest ecosystem in India

We studied for two years the seasonal changes in plant available nitrate and ammonium nitrogen (N), nitrification, N-mineralization, microbial biomass carbon (MBC), nitrogen (MBN) and phosphorus (MBP) in two forest and three cropland sites, derived from a tropical forest ecosystem of India. Results indicated that seasonal values of nitrate N, ammonium N and phosphate P ranged from 7.33–12.99, 5.1–10.22 and 4.0–7.8 μg g^?1 in forest and 4.13–9.26, 9.35–14.46 and 2.8–5.8 μg g^?1 in cropland ecosystems, respectively, with maximum values in summer and minimum in rainy seasons. Nitrification and N-mineralization values varied from 6–28 and 4–26 μg g^?1 mo^?1 in forest and 3–14 μg g^?1 mo^?1 and 4–17 μg g^?1 mo^?1 in cropland, with maximum values in rainy season and minimum in summer season.MBC, MBN MBP ranged from 393–753, 34–80 and 16–36 μg g^?1 in forests and 186–414, 21–41 and 11–22 μg g^?1 in croplands, being maximum in summer and minimum in rainy seasons. There was gradual increase in the values of inorganic N, nitrification, N-mineralization and MBC, MBN and MBP along the age of cropland. Analysis of variance indicated significant difference in the concentration of inorganic N, nitrification and N-mineralization and MBC, MBN and MBP due to sites and seasons.Cultivation caused decline in the mean annual organic C, N and P by 42%, 29% and 13%. The values of nitrate N were decreased by 23–38%, while ammonium N was increased by 39–74%. Nitrification and N-mineralization values were reduced by 39–63% and 40–60%, respectively. Microbial C, N and P were reduced by 44–54%, 41–50% and 28–44%, respectively. Nonetheless, the contribution of soil microbial biomass reflected in total N was enhanced from 4.76% in forest to 5.03% in cropland ecosystem. Enhancement of plant available ammonium-N and microbial contribution in total N are an indicator of natural conserving mechanism to check the nitrogen loss from the nutrient poor agro-ecosystem.     

Effect of polyethylene glycol,urea and sunflower meal on olive (Olea europaea var. europaea) leaf fermentation in continuous fermentors

A continuous culture system, inoculated with rumen liquor from goats or sheep, was used to study fermentation characteristics of olive leaves (OL). The effects of adding polyethylene glycol (PEG 4000 MW; 0, 2 or 20 g/100 g OL) and/or supplementing with urea (U) or sunflower meal (SM) (1.0 g N/100 g OM) were also studied. Olive leaf fermentation promoted low VFA production (35.2 mmol/d), predominantly of acetic acid, and low efficiency of VFA production (4.91 mol/kg digestible carbohydrates, DCHO). Both values increased with N supplementation, but effects of PEG were variable. No differences ascribed to the rumen inoculum origin were observed. The ammonia N concentration was increased only by supplementation with U. Total and amino acid N output was low and increased with N addition, but it was not affected by PEG treatment. No differences ascribed to the inoculum origin were observed concerning microbial N production rate or efficiency (g N/kg DCHO). There was no clear difference between sources of supplementary N regarding bacterial protein synthesis. On the basis of PEG results, the effect of tannins on OL fermentation was not important.     

Physicochemical and microbiological indicators of surface water body contamination with different sources of digestate from biogas plants

Transition from fossil energy sources to biogas production has resulted in a strong increase of leakage accidents from fermenters, but knowledge on the effects of fermentation product runoff into freshwater systems is currently restricted to direct toxicity due to oxygen depletion. This study provides first information about the influence of digestate runoff on the physicochemical habitat properties and the bacterial community composition of the hyporheic interstitial which is important in determining ecosystem functioning. We exposed natural stream beds to different concentrations of two different digestates from fermenters (corn and manure feedstock), hypothesizing that the digestate addition causes acute changes of the physicochemical parameters and has distinct effects on microbial community composition of the hyporheic interstitial depending on concentration and type of digestate. In line with the hypotheses, pH value, conductivity, redox potential and ammonium differed significantly from controls and among treatments after digestate addition, but only for a maximum of two days. pH values (controls: 7.8; corn: 7.9; manure: 7.9) and conductivity (controls: 813 μS/cm; corn: 969 μS/cm; manure: 1097 μS/cm) increased, the redox potential (controls: 153 mV; corn: 145 mV; manure: 144 mV) decreased the first two days. A high peak of ammonium-N was detected in the corn and manure treatments (controls: 5 mg/l, corn: 80 mg/l; manure: 60 mg/l) at day 1. In contrast, changes in bacterial community composition were detectable for longer periods of time (>5 days). Seventeen unique T-RF fingerprints of bacterial community response to each of the different digestate treatments (11 unique T-RFs in manure and 6 unique T-RFs in corn treatments) were found, suggesting that this approach provides a suitable ecological indicator for source tracking, e.g. in case of a biogas power plant leakage accident.     

Zinc protects Ceratophyllum demersum L. (free-floating hydrophyte) against reactive oxygen species induced by cadmium

Evidence for Zn protection against Cd-induced reactive oxygen species in the free-floating hydrophyte Ceratophyllum demersum L. is presented in this paper. Metal treatments of 10 μmol/L Cd, 10 Cd μmol/L supplemented with Zn (10, 50, 100 and 200 μmol/L) and Zn-alone treatments of the same concentrations were used. Using 5,5 dimethyl pyrroline-N-oxide as the spin-probe, electron spin resonance spectra indicated a drastic increase in hydroxyl radicals (OH) in Cd-10 μmol/L treatments, which was closely correlating with the enhanced formation of hydrogen peroxide (H₂O₂) and generation of superoxide radical (O₂^?) triggered by the oxidation of NADPH. The supplementation of adding Zn (10–200 μmol/L) to the Cd-10 μmol/L treatments significantly decreased the production of free radicals especially by eliminating the precursors of OH through inhibition of NADPH oxidation. Cd-enhanced ROS production which substantially increased the oxidative products of proteins measured as carbonyls was effectively inhibited by Zn supplementation.     

Effect of suplemental nitrogen source and feeding frequency on nutrient supply to lambs fed a kikuyu grass (Pennisetum clandestinum) hay-based diet

Eight castrated male lambs (35 ± 4 kg live weight), fed a basal diet of kikuyu grass hay, were used in a replicated 4 × 4 Latin Square experiment with a 2 × 2 factorial arrangement of treatments to evaluate the effect of supplemental feeding frequency and source of rumen degradable N on intake, digestibility, ruminal fermentation, and microbial protein yield. Treatments were supplementation with cassava meal plus calcium caseinate or cassava meal plus urea offered at a rate of 7 g/kg live weight daily in one or two meals per day. Lambs were fed twice daily in such manner to allow ad libitum comsumption of forage. There was significant feeding frequency by N source interaction on variables of intake. In general, intake of feed components was higher (P ≤ 0.05) by lambs offered the caseinate-supplement twice daily over intake observed in lambs given the others diet treatments. Digestibility of feed components was neither affected by supplemental N source (DM, P = 0.541; OM, P = 0.585; NDF, P = 0.828) nor by feeding frequency (DM, P = 0.122; OM, P = 0.175; NDF, P = 0.591). Urinary excretion of N increased (P ≤ 0.05) in lambs supplemented twice daily whereas N retention was similar for all treatments (N source, P = 0.748; feeding frequency, P = 0.418). Microbial protein entering into the small intestine was affected by the interaction between feeding frequency and N source such as an increasing (P < 0.10) in this variable was observed when lambs received the caseinate but not the urea supplement twice daily. Efficiency of microbial protein synthesis, however, was not affected by treatments (N source, P = 0.588; feeding frequency, P = 0.334). Rumen pH averaged 6.70 and it was neither affected by N source (P = 0.827) nor by feeding frequency (P = 0.740). Ruminal concentration of ammonia N was not affected by feeding frequency (P = 0.144) while it increased (P < 0.05) when urea rather than caseinate was the supplemental N source (mean of 7.61 mg/dl vs. 6.00 mg/dl). Concentration of sugars in rumen fluid was higher (P ≤ 0.05) in lambs supplemented once a day compared to twice daily (mean of 49.4 mg/dl vs. 34.4 mg/dl) for both N sources. A significant (P ≤ 0.05) N source by feeding frequency interaction effect was observed for ruminal concentrations of α-amino N compounds. In urea treatment α-amino N concentration increased (P ≤ 0.05) in lambs receiving the supplement twice daily compared to once a day (mean of 4.59 mg/dl vs. 3.70 mg/dl) while in caseinate treatment it was higher (P ≤ 0.05) in lambs offered the supplement in one meal per day compared to twice daily (mean of 5.29 mg/dl vs. 4.07 mg/dl). In conclusion, for ruminants fed a tropical grass-based diet, starch-rich supplement containing non-protein N as N source may be offered only once a day whereas the supply of nutrients may be improved if degradable true protein is included as N source and supplement is offered in two meals per day.